1. Field of the Invention
The present invention relates to a novel DNA encoding an enzyme which forms non-reducing saccharides having trehalose structure as an end unit from reducing amylaceous saccharides having a degree of glucose polymerization of 3 or higher, and a recombinant DNA and enzyme containing the DNA as well as to a transformant. The present invention further relates to preparations and uses thereof.
2. Description of the Prior Art
Trehalose is a disaccharide which consists of 2 glucose molecules that are linked together with their reducing groups, and, naturally, it is present in fungi, algae, insects, etc., in an extremely small quantity. Having no reducing residue within the molecule, trehalose does not cause an unsatisfactory browning reaction even when heated in the presence of amino acids or the like, and because of this it can sweeten food products without fear of causing unsatisfiable coloration and deterioration. Trehalose, however, is far from being readily prepared in a desired amount by conventional production methods, and, actually, it has rarely been used for sweetening food products.
Conventional production methods are roughly classified into 2 groups, i.e. the one using cells of microorganisms and the other employing a multi-enzymatic system wherein enzymes are allowed to act on saccharides. The former, as disclosed in Japanese Patent Laid-Open No. 154,485/75, is a method comprising growing microorganisms such as bacteria and yeasts in nutrient culture media, and collecting trehalose from the proliferated cells in the resultant cultures. The latter, as disclosed in Japanese Patent Laid-Open No. 216,695/83, is a method comprising providing maltose as a substrate, allowing a multi-enzymatic system using maltose- and trehalose-phosphorylases to act on maltose, and recovering the formed trehalose from the reaction system. Although the former facilitates growth of microorganisms with relative ease, it requires complex sequential complicated steps for collecting trehalose from the microorganisms containing only 15 w/w % trehalose, on a dry solid basis (d.s.b.). While the latter enables separating of trehalose with relative ease, but it is theoretically difficult to increase the trehalose yield by allowing enzymes to act on substrates at a considerably-high concentration because the enzymatic reaction in itself is an equilibrium reaction of 2 different types of enzymes and the equilibrium point constantly inclines to the side of forming glucose phosphate.
In view of the foregoing, the present inventors energetically screened enzymes which form saccharides having trehalose structure from amylaceous saccharides, and found that microorganisms such as those of the species Rhizobium sp. M-11 and Arthrobacter sp. Q36 produce a novel enzyme which forms non-reducing saccharides having trehalose structure as an end unit from reducing amylaceous saccharides having a degree of glucose polymerization of 3 or higher. Before or after this finding, it was revealed that such a non-reducing saccharide is almost quantitatively hydrolyzed into trehalose and glucose and/or maltooligosaccharides by another enzyme produced by the same microorganisms as mentioned above. Since using a combination use of these enzymes enables the formation of a desired amount of trehalose with relative ease, the aforementioned objects relating to trehalose would be completely overcome. The low level production of the novel enzyme by such a microorganism, i.e. in a relatively-large scale culture present a drawback in industrially producing trehalose and/or non-reducing saccharides having trehalose structure as an end unit.
Recombinant DNA technology has made a remarkable progress in recent years. At present, even an enzyme whose total amino acid sequence has not been revealed can be readily prepared in a desired amount, if a gene encoding the enzyme was once isolated and the base sequence was decoded, by preparing a recombinant DNA which contains a DNA encoding the enzyme, introducing the recombinant DNA into microorganisms or cells of plants and animals, and culturing the resultant transformants. With this background, a gene encoding the enzyme and its base sequence we urgently sought.